Viral replication in tumor cells leads to their destruction, with release of progeny virion that infect adjacent cancer cells in a process referred to as viral oncolysis. Clinical trials of oncolytic viruses have demonstrated both safety and potential efficacy of this approach. We have conducted research in the prior funding period demonstrating the utility of Herpes simplex virus 1 (HSV-1) mutants for viral oncolysis of liver tumors. We characterized regulation of HSV-1 replication at the molecular level, and used this information to 1) genetically engineer HSV-1 mutants that when introduced intravascularly into the liver replicate preferentially in liver metastases rather than normal cells; 2) express therapeutic transgenes such as prodrug activation genes (e.g.cytosine deaminase); 3) enhance survival of animals with diffuse liver tumors following intravascular delivery. Intratumoral conversion of 5-FC to 5-FU by the yeast cytosine deaminase transgene when added to HSV-1 oncolysis enhances efficacy. We have taken the most significant laboratory findings and moved them into clinical trials for patients with liver tumors with the assistance of the NCI RapidAccess to Intervention Development (RAID) program. We propose projects for this competing renewal application that expand on findings and observations made during the previous funding period. We propose to elucidate mechanisms of interaction between prodrug bioactivation and HSV-1 oncolysis. Positron emission tomography (PET) has been used to examine gene expression; however, we will develop PET to quantitatively assess viral replication. Presently, detection of viral replication requires sampling (biopsy) of tissues for molecular analyses. Because these techniques are invasive, cumbersome, and non-quantitative, they are not useful in human clinical trials of HSV-1 oncolysis. Accordingly there exists a strong rationale to develop minimally invasive imaging methods to quantitatively assess sites of HSV-1 replication. Successful development of quantitative imaging of viral replication will improve the quality of both preclinical and clinical studies of HSV-1 oncolysis. In Specific Aim 1, we will define interactions between intracellular conversion of 5-FC to 5-FU via the yeast cytosine deaminase transgene, HSV-1 replication, and anti-neoplastic efficacy. In Specific Aim 2, we will compare the dopamine receptor and thymidine kinase enzyme as reporters to quantitatively image viral replication with PET. We will co-register quantitative PET imaging of viral replication with imaging of tumor metabolic activity to simultaneously assess viral replication and anti-neoplastic response. We will also use [19F] magnetic resonance spectroscopy (MRS) to image cytosine deaminase transgene function. We are in position to directly translate observations from these experiments into clinical trials.